Brefeldin A (BFA) is a drug that inhibits ARF activation by three mammalian guanine nucleotide-exchange proteins and thereby blocks initiation of transport vesicle formation at specific intracellular sites. BIG1 and BIG2 act at trans-Golgi membranes or later in the secretory pathway, whereas GBF1 predominately associated with cis-Golgi at an earlier stage. Pertubation of endoplasmic reticulum (ER) functions can result in accumulation of unfolded or misfolded proteins that causes ER stress and unfolded protein response (UPR), with accumulation of ER stress response element (ERSE) gene products. BFA treatment of cells causes accumulation of proteins in the ER, ER stress, and ultimately apoptosis. To assess involvement of BFA-sensitive GEFs in the damage resulting from prolonged BFA treatment, HepG2 cells were selectively depleted of BIG1, BIG2, or GBF1 by using specific siRNA. Only GBF1 siRNA dramatically slowed cell growth, led to cell-cycle arrest in Go/G1 phase, and caused dispersion of Golgi markers beta-COP and GM130, whereas ER structure appeared intact. GBF1 depletion also significantly increased levels of ER proteins calreticulin and protein disulfide isomerase (PDI). Proteomic analysis identified ER chaperones involved in the UPR that were significantly increased in amounts in BGF1-depleted cells. Upon ER stress, transcription factor ATF6 translocates from the ER to Golgi, where it is sequentially cleaved by site 1 and site 2 proteases, S1P and S2P, to a 50-kDa form that activates transcription of ERSE genes. Depletion of GBF1, but not BIG1 or BIG2 induced relocation of S2P from Golgi to ER with proteolysis of ATF6 followed by up-regulation of ER chaperones, mimicking a UPR response. Because BIG1 and BIG2, the only other BFA-inhibited GEPs in mammalian cells are responsible for ARF activation at sites distal to that of entry of new proteins to the trafficking system, their absence may not cause ER stress as quickly as does GBF1 depletion.[unreadable] [unreadable] Our earlier report (Padilla et al., 2004) that BIG1 accumulated in nucleoli of serum-deprived HepG2 cells prompted us to identify molecules associated with it in dynamic nucleolar structures. Antibodies against BIG1 or nucleolin coprecipitated both proteins from nuclei, which was abolished by the incubation of nuclei with RNase A or DNase, indicating that the interaction depended on nucleic acids. 32P labeling of RNAs immunoprecipitated from nuclei with BIG1 or nucleolin revealed bands of approx. 210 bases that also hybridized with U3 small nucleolar (sno)RNA-specific oligonucleotides. Clones of U3 snoRNA cDNAs from the material precipitated by antibodies against BIG1 or nucleolin yielded identical nucleotide sequences that also were found in genomic DNA. Later analyses revealed the presence of fibrillarin, nucleoporin p62, and La in BIG1 and nucleolin immunoprecipitates. Our data demonstrte that BIG1, nucleolin, U3, the U3-binding protein fibrillarin, and the RNA-binding protein La may act together in nuclear complexes, consistent with a potential role for BIG1 in nucleolar processes. Evidence that BIG1 and nucleolin, but not fibrillarin, can be present with p62 at the nuclear envelope confirms the presence of BIG1 and nucleolin in dynamic molecular complexes that change in composition while moving through nuclei. Nuclear functions of BIG1 remain to be determined.